α fetoprotein

Alkaline phosphatase staining was performed using a Fast Red substrate kit (Nichirei Biosciences Inc., Tokyo, Japan). To detect pluripotent stem cell marker antigens cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the cells were treated with PBS containing 5% normal goat serum (Nichirei) and 0.1% Triton X-100 for 45 min at room temperature. Fixed cells were stained with primary antibodies included SSEA-4 (1:100, Stemgent®, Cambridge, MA), TRA-1-60 (1/200, Stemgent®), TRA-1-81 (1/200, Stemgent®), Oct-3/4 (1/200 Millipore), Nanog (1/600, ReproCELL, Yokohama, Japan), Nestin (1/200, Millipore), βIII-tubulin (1/200, Millipore), α-smooth muscle actin (pre-diluted, DAKO Cytomation, Glostrup, Denmark) and α-fetoprotein (1/100, R&D Systems). These primary antibodies were visualized with Alexa Fluor® 488- conjugated goat anti-rabbit IgG, or Alexa Fluor® 594-conjugated goat anti-rabbit IgG, or Alexa Fluor® 488-conjugated goat anti-mouse IgG, or Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/200, Invitrogen, Carlsbad, CA). Nucleuses were stained with DAPI. Fluorescence images were acquired using a Zeiss inverted LSM confocal microscope (Carl Zeiss, GmbH, Germany).

Cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature and washed with PBS. Continuously, cells were treated with PBS containing 5% normal goat or rabbit serum (Nichirei Biosciences Inc., Tokyo, Japan) and 0.1% Triton X-100 at room temperature. Then cells were fixed and stained with antibodies to Oct4 (1/200 Millipore, Billerica, MA), Tra-1-60 (1/200, Stemgent^®, Cambridge, MA), Tra-1-81 (1/200, Stemgent^®), SSEA-1 (1/100, Stemgent^®), SSEA-4 (1/100, Stemgent^®), MAP2 (1/200, Millipore), Nestin (1/200, Millipore), α-smooth muscle actin (α-SMA: pre-diluted, DAKO Cytomation, Glostrup, Denmark), α-fetoprotein (1/100, R&D Systems Minneapolis, MN), and to the SeV nucleocapsid protein (mouse monoclonal antibody, clone #2E4, 1.6 mg/mL). These primary antibodies were visualized with Alexa Fluor^® 488-conjugated goat anti-rabbit IgG, or Alexa Fluor^® 488-conjugated rabbit anti-mouse IgG, or Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/200, Invitrogen, Carlsbad, CA). Cell nuclei were stained with 4', 6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Fluorescence images were taken using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss, GmbH, Germany). Alkaline phosphate (ALP) staining was performed using a Fast Red substrate kit (Nichirei) according to manufacturer’s instruction.